ROLIMUS SERIES

rolimus series

mTOR inhibitor
temsirolimus (CCI-779), everolimus (RAD001), deforolimus (AP23573), AP21967, biolimus, AP23102, zotarolimus (ABT 578), sirolimus (Rapamune), and tacrolimus (Prograf).


1 SIROLIMUS
2 TEMSIROLIMUS
3 EVEROLIMUS
4 RIDAFOROLIMUS
5 ZOTAROLIMUS
6 BIOLIMUS
7 PIMECROLIMUS
8TACROLIMUS
9



WILL BE UPDATED WATCH OUT


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DR ANTHONY CRASTO 
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1 SIROLIMUS (RAPAMYCIN)


File:Sirolimus.svg
Rapamycin (Sirolimus)
(3S,6R,7E,9R,10R,12R,14S,15E,17E,19​E,21S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,​25, 26,27,32,33,34,34a-Hexadecahydro-9,27-dihydroxy-3-​[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]​-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-he​xamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]oxaazacy​clohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone
Wyeth Pharmaceuticals (Originator)
M.Wt:914.18
Formula:C51H79NO13
53123-88-9 cas no
Antifungal and immunosuppressant. Specific inhibitor of mTOR (mammalian target of Rapamycin). Complexes with FKBP-12 and binds mTOR inhibiting its activity. Inhibits interleukin-2-induced phosphorylation and activation of p70 S6 kinase. Induces autophagy in yeast and mammalian cell lines.
Rapamycin is a triene macrolide antibiotic, which demonstrates anti-fungal, anti-inflammatory, anti-tumor and immunosuppressive properties. Rapamycin has been shown to block T-cell activation and proliferation, as well as, the activation of p70 S6 kinase and exhibits strong binding to FK-506 binding proteins. Rapamycin also inhibits the activity of the protein, mTOR, (mammalian target of rapamycin) which functions in a signaling pathway to promote tumor growth. Rapamycin binds to a receptor protein (FKBP12) and the rapamycin/FKB12 complex then binds to mTOR and prevents interaction of mTOR with target proteins in this signaling pathway. Rapamycin name is derived from the native word for Easter Island, Rapi Nui.
  • (-)-Rapamycin
  • Antibiotic AY 22989
  • AY 22989
  • AY-22989
  • CCRIS 9024
  • HSDB 7284
  • NSC 226080
  • Rapammune
  • Rapamune
  • Rapamycin
  • SILA 9268A
  • Sirolimus
  • UNII-W36ZG6FT64
  • WY-090217
  • A 8167
A macrolide compound obtained from Streptomyces hygroscopicus that acts by selectively blocking the transcriptional activation of cytokines thereby inhibiting cytokine production. It is bioactive only when bound to IMMUNOPHILINS. Sirolimus is a potent immunosuppressant and possesses both antifungal and antineoplastic properties.

Sirolimus (INN/USAN), also known as rapamycin, is an immunosuppressant drug used to prevent rejection in organ transplantation; it is especially useful in kidney transplants. It prevents activation of T cells and B cells by inhibiting their response to interleukin-2 (IL-2). Sirolimus is also used as a coronary stent coating. Sirolimus works, in part, by eliminating old and abnormal white blood cells.[citation needed] Sirolimus is effective in mice with autoimmunity and in children with a rare condition called autoimmune lymphoproliferative syndrome (ALPS).
sirolimus
macrolide, sirolimus was discovered by Brazilian researchers as a product of the bacterium Streptomyces hygroscopicus in a soil sample fromEaster Island[1] — an island also known as Rapa Nui.[2] It was approved by the FDA in September 1999 and is marketed under the trade nameRapamune by Pfizer (formerly by Wyeth).
Sirolimus was originally developed as an antifungal agent. However, this use was abandoned when it was discovered to have potent immunosuppressive and antiproliferative properties. It has since been shown to prolong the life of mice and might also be useful in the treatment of certain cancers.
Unlike the similarly named tacrolimus, sirolimus is not a calcineurin inhibitor, but it has a similar suppressive effect on the immune system. Sirolimus inhibits the response tointerleukin-2 (IL-2), and thereby blocks activation of T and B cells. In contrast, tacrolimus inhibits the secretion of IL-2.
The mode of action of sirolimus is to bind the cytosolic protein FK-binding protein 12(FKBP12) in a manner similar to tacrolimus. Unlike the tacrolimus-FKBP12 complex which inhibits calcineurin (PP2B), the sirolimus-FKBP12 complex inhibits themammalian target of rapamycin (mTOR, rapamycin being an older name for sirolimus) pathway by directly binding the mTOR Complex1 (mTORC1).
mTOR has also been called FRAP (FKBP-rapamycin associated protein), RAFT (rapamycin and FKBP target), RAPT1, or SEP. The earlier names FRAP and RAFT were coined to reflect the fact that sirolimus must bind FKBP12 first, and only the FKBP12-sirolimus complex can bind mTOR. However, mTOR is now the widely accepted name, since Tor was first discovered via genetic and molecular studies of sirolimus-resistant mutants of Saccharomyces cerevisiae that identified FKBP12, Tor1, and Tor2 as the targets of sirolimus and provided robust support that the FKBP12-sirolimus complex binds to and inhibits Tor1 and Tor2.
rapamycin
Unlike the similarly named tacrolimus, sirolimus is not a calcineurin inhibitor, but it has a similar suppressive effect on the immune system. Sirolimus inhibits the response to interleukin-2 (IL-2), and thereby blocks activation of T and B cells. In contrast, tacrolimus inhibits the secretion of IL-2.
The mode of action of sirolimus is to bind the cytosolic protein FK-binding protein 12 (FKBP12) in a manner similar to tacrolimus. Unlike the tacrolimus-FKBP12 complex which inhibits calcineurin (PP2B), the sirolimus-FKBP12 complex inhibits the mammalian target of rapamycin(mTOR, rapamycin being an older name for sirolimus) pathway by directly binding the mTOR Complex1 (mTORC1).
mTOR has also been called FRAP (FKBP-rapamycin associated protein), RAFT (rapamycin and FKBP target), RAPT1, or SEP. The earlier names FRAP and RAFT were coined to reflect the fact that sirolimus must bind FKBP12 first, and only the FKBP12-sirolimus complex can bind mTOR. However, mTOR is now the widely accepted name, since Tor was first discovered via genetic and molecular studies of sirolimus-resistant mutants of Saccharomyces cerevisiae that identified FKBP12, Tor1, and Tor2 as the targets of sirolimus and provided robust support that the FKBP12-sirolimus complex binds to and inhibits Tor1 and Tor2.
SIROLIMUS




Rapamycin and its preparation are described in US Patent No. 3,929,992, issued December 30, 1975. Alternatively, rapamycin may be purchased commercially [Rapamune®, Wyeth].


Rapamycin (Sirolimus) is a 31-member natural macrocyclic lactone [C51H79N1O13; MWt=914.2] produced by Streptomyces hygroscopicus and found in the 1970s (U.S. Pat. No. 3,929,992; 3,993,749). Rapamycin (structure shown below) was approved by the Food and Drug Administration (FDA) for the prophylaxis of renal transplant rejection in 1999.

Figure US08088789-20120103-C00001

Rapamycin resembles tacrolimus (binds to the same intracellular binding protein or immunophilin known as FKBP-12) but differs in its mechanism of action. Whereas tacrolimus and cyclosporine inhibit T-cell activation by blocking lymphokine (e.g., IL2) gene transcription, sirolimus inhibits T-cell activation and T lymphocyte proliferation by binding to mammalian target of rapamycin (mTOR). Rapamycin can act in synergy with cyclosporine or tacrolimus in suppressing the immune system.
Rapamycin is also useful in preventing or treating systemic lupus erythematosus [U.S. Pat. No. 5,078,999], pulmonary inflammation [U.S. Pat. No. 5,080,899], insulin dependent diabetes mellitus [U.S. Pat. No. 5,321,009], skin disorders, such as psoriasis [U.S. Pat. No. 5,286,730], bowel disorders [U.S. Pat. No. 5,286,731], smooth muscle cell proliferation and intimal thickening following vascular injury [U.S. Pat. Nos. 5,288,711 and 5,516,781], adult T-cell leukemia/lymphoma [European Patent Application 525,960 A1], ocular inflammation [U.S. Pat. No. 5,387,589], malignant carcinomas [U.S. Pat. No. 5,206,018], cardiac inflammatory disease [U.S. Pat. No. 5,496,832], anemia [U.S. Pat. No. 5,561,138] and increase neurite outgrowth [Parker, E. M. et al, Neuropharmacology 39, 1913-1919, 2000].
Although rapamycin can be used to treat various disease conditions, the utility of the compound as a pharmaceutical drug has been limited by its very low and variable bioavailability and its high immunosuppressive potency and potential high toxicity. Also, rapamycin is only very slightly soluble in water. To overcome these problems, prodrugs and analogues of the compound have been synthesized. Water soluble prodrugs prepared by derivatizing rapamycin positions 31 and 42 (formerly positions 28 and 40) of the rapamycin structure to form glycinate, propionate, and pyrrolidino butyrate prodrugs have been described (U.S. Pat. No. 4,650,803). Some of the analogues of rapamycin described in the art include monoacyl and diacyl analogues (U.S. Pat. No. 4,316,885), acetal analogues (U.S. Pat. No. 5,151,413), silyl ethers (U.S. Pat. No. 5,120,842), hydroxyesters (U.S. Pat. No. 5,362,718), as well as alkyl, aryl, alkenyl, and alkynyl analogues (U.S. Pat. Nos. 5,665,772; 5,258,389; 6,384,046; WO 97/35575).


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Synthesis
PREPARATION
CUT PASTE FROM TEXT
In one embodiment of this invention rapamycin is prepared in the followingmanner: 4
A suitable fermenter is charged with production meis reached in the fermentation mixture after 2-8 days,
usually after about 5 days, as determined by the cup plate method and Candida albicans as the test organism. The mycelium is harvested by filtration with diatomaceous earth. Rapamycin is then extracted from the mycelium with a water-miscible solvent, for example a lower alkanol, preferably methanol or ethanol. The latter extract is then concentrated, preferably under reduced pressure, and the resulting aqueous phase is extracted with a water-immiscible solvent. A preferred water-immiscible solvent for this purpose is methylene dichloride although chloroform, carbon tetrachloride, benzene, n-butanol and the like may also be used. The latter extract is concentrated, preferably under reduced pressure, to afford the crude product as an oil.
The product may be purified further by a variety of methods. Among the preferred methods of purification is to dissolve the crude product in a substantially nonpolar, first solvent, for example petroleum ether or hexane, and to treat the resulting solution with a suit able absorbent, for example charcoal or silica gel, so that the antibiotic becomes absorbed on the absorbant. The absorbant is then separated and washed or eluted with a second solvent more polar than the first solvent, for example ethyl acetate, methylene dichloride, or a mixture of methylene dichloride and ether (preferred). Thereafter, concentration of the wash solution or eluate affords substantially pure rapamycin. Further purification is obtained by partial precipitation with a nonpolar solvent, for example, petroleum ether, hexane, pentane and the like, from a solution of the rapamycin in a more polar solvent, for example, ether, ethyl acetate, benzene and the like. Still-further purification is obtained by column chromatography, preferably employing silica gel, and by crystallization of the rapamycin from ether.
In another preferred embodiment of this invention a first stage inoculum of S treptomyces hygroscopicus NRRL 5491 is prepared in small batches in a medium containing soybean flour, glucose, ammonium sulfate, and calcium carbonate incubated at about 25C at pH 7.l-7.3 for 24 hrs. with agitation, preferably on a gyrotary shaker. The growth thus obtained is used to inoculate a number of somewhat larger batches of the same medium as described above which are incubated at about 25C and pH 7.1-7.3 for 18 hrs. with agitation, preferably on a reciprocating'shaker, to obtain a sec- "ond stagc inoculum which is used to inoculate the production stage fermenters.
6 5.86'.2.-The fermenters are inoculated with the second stage inoculum described above and incubated at about 25C with' agitationand aeration while controlling and 'mai'ntaining the mixture at approximately pH 6.0 by
addition offa base, for example, sodium hydroxide, potassium hydroxide or preferably ammonium hydroxide, as required from time to time. Addition of a source -of assimilable carbon, preferably glucose, is started when theconcentrationof the latter in the broth has dropped to about 0.5% wt/vol, normally about 48 hrs after. the start of fermentation, and is maintained until the end ofthe particular run. In this manner a fermentation broth containing about 60 ug/ml of rapamycin as determined by the assay method described above is obtained in 45 days, when fermentation is stopped.
' Filtration of the'mycelium, mixing the latter with a watef-miscible 'lower' alkanol, preferably methanol, followed by extraction with a halogenated aliphatic hydrocarbon, preferably trichloroethane, and evaporation of the solvents yields a first oily residue. This first oily residue is dissolved in a lower aliphatic ketone, preferably acetone, filtered from insoluble impurities, the filtrate evaporated to yield a second oily residue which is extractedjwith a water-miscible lower alkanol,
preferably methanol, and the latter extract is evaporated to yield crude rapamycin as a third oily residue. This third oily residue is dissolved in a mixture of a lower aliphatic ketone and a lower aliphatic hydrocarbon, preferably acetone-hexane, an absorbent such as charcoal or preferably silica gel is added to adsorb the rapamycin, the latter is eluted from the adsorbate with a similar but more polar solvent mixture, for example a mixture as above but containing a higher proportion of the aliphatic ketone, the eluates are evaporated and the residue is crystallized from diethyl ether, to yield pure crystalline rapamycin. In this manner a total of 45-5 8% of the rapamycin initially present in the fermentation mixture is recovered as pure crystalline rapamycin.
CHARACTERIZATION solvent systems; for example, ether-hexane 40:60 (Rf 0.42), 'isopropyl alcoholvbenzene 15:85 (Rf= 0.5) and ethanol-benzene 20:80 (Rf f 0.43);
d. rapamycin obtained from four successive fermentation batchesgave the following values on repeated The production stage fermenters are equipped with 7 devices for controlling and maintaining pH at a predetermined level and for continuous metered addition of elemental analyses:
AVER- e. rapamycin exhibits the following characteristic absorption maxima in its ultraviolet absorption spectrum ethanol):
f. the infrared absorption spectrum of rapamycin in chloroform is reproduced in FIG. 1 and shows characteristic absorption bands at 3560, 3430, 1730, 1705 and 1630-1610 cm;
Further infrared absorption bands are characterized by the following data given in reciprocal centimeters with (s) denoting a strong, (m) denoting a medium, and (w) denoting a weak intensity band. This classification is arbitrarily selected in such a manner that a band is denoted as strong (s) if its peak absorption is more than two-thirds of the background in the same region; medium (m) if its peak is between one-third and twothirds of the background in the same region; and weak (w) if its peak is less than one-third of the background in the same region.
2990 cm (m) 1158 cm" (m) 2955 cm (s) 1129 cm (s) 2919 cm (s) 1080 cm (s) 2858 cm (s) 1060 cm (s) 2815 cm (m) 1040 cm (m) 1440 cm (s) 1020 crn' (m) 1365 cm (m) 978 cm" (s) 1316 cm (in) 905 cm (m) 1272 cm (m) 888 cm" (w) 1178 cm (s) 866 cm- (w) g. the nuclear magnetic resonance spectrum of rapamycinin deuterochloroform is reproduced in FIG. 2; SEE PATENT
CLAIMS
l. Rapamycin, an antibiotic which a. is a colourless, crystalline compound with a melting point of 183 to l8SC, after recrystallization from ether;
b. is soluble in ether, chloroform, acetone, methanol and dimethylformamide, very sparingly soluble in hexane and petroleum ether and substantially insoluble in water;
c. shows a uniform spot on thin layer plates of silica gel",
d. has a characteristic elemental analysis of about C,
e. exhibits the following characteristic absorption maxima in its ultraviolet absorption spectrum (95% ff has 'a characteristic infrared absorption spectrum shown in accompanying FIG. 1; SEE PATENT
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Rapamycin synthetic studies. 1. Construction of the C(27)-C(42) subunit. Tetrahedron Lett 1994, 35, 28, 4907


A partial synthesis of rapamycin has been reported: The condensation of sulfone (I) with epoxide (II) by means of butyllithium followed by desulfonation with Na/Hg gives the partially protected diol (III), which is treated with methanesulfonyl chloride and NaH to afford the epoxide (IV). Ring opening of epoxide (IV) with LiI and BF3.Et2O followed by protection of the resulting alcohol with PMBOC(NH)CCl3 yields the primary iodo compound (V). The condensation of (V) with the fully protected dihydroxyaldehyde (VI) (see later) by means of butyllithium in THF/HMPT gives the fully protected trihydroxyketone (VII), which is hydrolyzed with camphorsulfonic acid (CSA) to the corresponding gemdiol and reprotected with pivaloyl chloride (the primary alcohol) and tert-butyldimethylsilyl trifluoromethanesulfonate (the secondary alcohol), yielding a new fully protected trihydroxyketone (VIII). Elimination of the pivaloyl group with DIBAL and the dithiane group with MeI/CaCO3 affords the hydroxyketone (IX), which is finally oxidized with oxalyl chloride to the ketoaldehyde (X), the C(27)-C(42) fragment [the C(12)-C(15) fragment with the C(12)-substituent based on the IUPAC nomenclature recommendations]. The fully protected dihydroxyaldehyde (VI) is obtained as follows: The reaction of methyl 3-hydroxy-2(R)-methylpropionate (XI) with BPSCl followed by reduction with LiBH4 to the corresponding alcohol and oxidation with oxalyl chloride gives the aldehyde (XII), which is protected with propane-1,3-dithiol and BF3.Et2O to afford the dithiane compound (XIII). Elimination of the silyl group with TBAF followed by esterification with tosyl chloride, reaction with NaI and, finally, with sodium phenylsulfinate gives the sulfone (XIV), which is condensed with the partially protected dihydroxyaldehyde (XV), oxidized with oxalyl chloride and desulfonated with Al/Hg to afford the dithianyl ketone (XVI). The reaction of (XVI) with lithium hexamethyldisilylazane gives the corresponding enolate, which is treated with dimethyllithium cuprate to yield the fully protected unsaturated dihydroxyaldehyde (VI).


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The Ley Synthesis of Rapamycin

Rapamycin (3) is used clinically as an immunosuppressive agent. The synthesis of 3 (Angew. Chem. Int. Ed. 200746, 591. DOI: 10.1002/anie.200604053) by Steven V. Ley of the University of Cambridge was based on the assembly and subsequent coupling of the iododiene 1 and the stannyl alkene 2.
The lactone of 1 was prepared by Fe-mediated cyclocarbonylation of the alkenyl epoxide 5, following the protocol developed in the Ley group.
The cyclohexane of 2 was constructed by SnCl4-mediated cyclization of the allyl stannane 9, again employing a procedure developed in the Ley group. Hydroboration delivered the aldehyde 11, which was crotylated with 12, following the H. C. Brown method. The alcohol so produced (not illustrated) was used to direct the diastereoselectivity of epoxidation, then removed, to give 13. Coupling with 14 then led to 2.
Combination of 1 with 2 led to 15, which was condensed with catechol to give the macrocycle 16. Exposure of 16 to base effected Dieckmann cyclization, to deliver the ring-contracted macrolactone 17, which was carried on to (-)-rapamycin (3).

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Total Synthesis of Rapamycin

Angewandte Chemie International Edition

Volume 46, Issue 4, pages 591–597, January 15, 2007
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rapamycin_1.jpg
Ley, Maddess, Tackett, Watanabe, Brennan, Spilling, Scott and Osborn. ACIEE2006EarlyView. DOI:10.1002/anie.200604053.
It’s been in the works for quite a while, but Steve Ley’s synthesis of Rapamycin has just been published. This complex beast has a multitude of biological activities, including an interesting immunosuppressive profile, resulting in clinical usage following organ transplantation. So, unsurprisingly, it’s been the target of many projects, with complete total syntheses published by SmithDanishefskySchreiber and KCN.
So what makes this one different? Well, it does have one of the most interesting macrocyclisations I’ve seen since Jamison’s paper, and a very nice demonstration of the BDA-aldol methodology. The overall strategy is also impressive, so on with the retro:
rapamycin_2.jpg
First stop is the BDA-aldol; this type of chemistry is interesting, because the protecting group for the diol is also the stereo-directing group. The stereochemistry for this comes from a glycolic acid, and has been usedin this manner by the group before. The result is as impressive as ever, with a high yield, and presumably a very high d.r. (no mention of actual numbers).
rapamycin_3.jpg
The rest of the fragment synthesis was completed in a succinct and competent manner, but using relatively well known chemistry. However, I was especially impressed with the macrocyclisation I mentioned:
rapamycin_4.jpg
Tethering the free ends of the linear precursor with a simple etherification/esterification onto catechol gave then a macrocycle holding the desired reaction centres together. Treatment of this with base then induces a Dieckmann-condensation type cyclisation to deliver the desired macrocycle. Of course, at this stage, only a few more steps were required to complete the molecule, and end an era of the Wiffen Lab.
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Drugs Fut 1999, 24(1): 22
DOI: 10.1358/dof.1999.024.01.474036


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BMC Plant Biol. 2007 Jun 1;7:26.

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Maddess ML, Tackett MN, Watanabe H, Brennan PE, Spilling CD, Scott JS, Osborn DP, Ley SV.
Angew Chem Int Ed Engl. 2007;46(4):591-7. No abstract available.

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21 Fleming et al (2011) Chemical modulators of autophagy as biological probes and potential therapeutics. 7 9. PMID:21164513.

22 J Am Chem Soc1993,115,(10):4419

23 Tetrahedron Lett1994,35,(28):4911
24 Chemistry (Weinheim)1995,1,(5):318

24
Figure imgf000004_0001SIROLIMUS

FEMALE FERTILITY
http://amcrasto.theeurekamoments.com/2013/02/11/immunosuppressant-drug-rapamycin-helps-preserving-female-fertility/

PATENTS
Canada2293793APPROVED2006-07-11EXP    2018-06-11
Canada2103571                2003-04-29          2012-02-21
United States5989591                1998-09-11          2018-09-11
United States5212155                1993-05-18          2010-05-18


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A plaque, written in Brazilian Portuguese, commemorating the discovery of sirolimus on Easter Island, near Rano Kau


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DR ANTHONY CRASTO 
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2   TEMSIROLIMUS
TEMSIROLIMUS
Proline CCI-779
Torisel, NCGC00167518-01
LAUNCHED 2007
PFIZER
  • CCI 779
  • CCI-779
  • HSDB 7931
  • Temsirolimus
  • Torisel
  • UNII-624KN6GM2T
  • WAY-CCI 779
Inhibits mTOR protein
For the treatment of renal cell carcinoma (RCC). Also investigated for use/treatment in breast cancer, lymphoma (unspecified), rheumatoid arthritis, and multiple myeloma.
An ester analog of rapamycin. Temsirolimus binds to and inhibits the mammalian target of rapamycin (mTOR), resulting in decreased expression of mRNAs necessary for cell cycle progression and arresting cells in the G1 phase of the cell cycle. mTOR is a serine/threonine kinase which plays a role in the PI3K/AKT pathway that is upregulated in some tumors
(1R,2R,4S)-4-{(2R)-2-[(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5,6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-tetracosahydro-3H-23,27-epoxypyrido[2,1-c][1,4]oxazacyclohentriacontin-3-yl]propyl}-2-methoxycyclohexyl 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate
cas 162635-04-3 
Temsirolimus is an intravenous drug for the treatment of renal cell carcinoma (RCC), developed by Wyeth Pharmaceuticals and approved by the FDA in late May 2007, and was also approved by the European Medicines Agency (EMEA) on November 2007. It is a derivative of sirolimus and is sold as Torisel.
Molecular Formula: C56H87NO16
Molecular Weight: 1030.28708
Temsirolimus (CCI-779) is an intravenous drug for the treatment of renal cell carcinoma (RCC), developed by WyethPharmaceuticals and approved by the U.S. Food and Drug Administration (FDA) in late May 2007, and was also approved by the European Medicines Agency (EMEA) on November 2007. It is a derivative of sirolimus and is sold as Torisel.
TEMSIROLIMUS

Temsirolimus is a specific inhibitor of mTOR and interferes with the synthesis of proteins that regulate proliferation, growth, and survival of tumor cells. Treatment with temsirolimus leads to cell cycle arrest in the G1 phase, and also inhibits tumor angiogenesis by reducing synthesis of VEGF.
The product had been under development by Wyeth Pharmaceutical for the treatment of pancreas cancer and metastatic breast cancer, multiple sclerosis (MS) and rheumatoid arthritis (RA); however, no recent development for these indications has been reported. Pfizer had been developing the compound for the treatment of sarcoma.
Temsirolimus holds orphan drug designation in both the U.S. and the E.U. for the treatment of renal cell carcinoma. Orphan drug designation was received in the U.S. in 2006 for the treatment of mantle-cell lymphoma.
mTOR (mammalian target of rapamycin) is a kinase enzyme inside the cell that collects and interprets the numerous and varied growth and survival signals received by tumor cells. When the kinase activity of mTOR is activated, its downstream effectors, the synthesis of cell cycle proteins such as cyclin D and hypoxia-inducible factor-1a (HIF-1a) are increased. HIF-1a then stimulates VEGF. Whether or not mTOR kinase is activated, determines whether the tumor cell produces key proteins needed for proliferation, growth, survival, and angiogenesis.
mTOR is activated in tumor cells by various mechanisms including growth factor surface receptor tyrosine kinases, oncogenes, and loss of tumor suppressor genes. These activating factors are known to be important for malignant transformation and progression.mTOR is particularly important in the biology of renal cancer (RCC) owing to its function in regulating HIF-1a levels. Mutation or loss of the von Hippel Lindau tumor-suppressor gene is common in RCC and is manifested by reduced degradation of HIF-1a. In RCC tumors, activated mTOR further exacerbates accumulation of HIF-1a by increasing synthesis of this transcription factor and its angiogenic target gene products.
Rapamycin 42-ester with 3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid (CCl-779) is an ester of rapamycin which has demonstrated significant inhibitory effects on tumor growth in both in vitro and in vivo models.
CCl-779 may delay the time to progression of tumors or time to tumor recurrence which is more typical of cytostatic rather than cytotoxic agents. CCl-779 is considered to have a mechanism of action that is similar to that of sirolimus. CCl-779 binds to and forms a complex with the cytoplasmic protein FKBP, which inhibits an enzyme, mTOR (mammalian target of rapamycin, also known as FKBP12-rapamycin associated protein [FRAP]). Inhibition of mTOR's kinase activity inhibits a variety of signal transduction pathways, including cytokine-stimulated cell proliferation, translation of mRNAs for several key proteins that regulate the G1 phase of the cell cycle, and IL-2-induced transcription, leading to inhibition of progression of the cell cycle from G1 to S. The mechanism of action of CCl-779 that results in the G1-S phase block is novel for an anticancer drug.
The preparation and use of hydroxyesters of rapamycin, including CCl-779, are disclosed in U.S. Pat. No. 5,362,718. A regiospecific synthesis of CCl-779 is described in U.S. Pat. No. 6,277,983.
CCl-779 can be synthesized by the non-regioselective acylation of rapamycin, as described in U.S. Pat. No. 5,362,718. The synthesis, however, is complicated by mixtures of the desired 42-ester, with 31-esterified rapamycin, as well as 31, 42-diesterified rapamycin and unreacted rapamycin.
CCl-779 can also be prepared by the acylation of the 31-silyl ether of rapamycin with a ketal of bis-(hydroxymethyl)propionic acid, followed by removal of the 31-silyl ether and ketal protecting group from the bis-(hydroxymethyl) propionic acid, as described in U.S. Pat. No. 6,277,983. However, the crude 42-monoester produced from this regioselective synthesis requires further purification by column chromatography to remove residual amounts of diester by-products and unreacted rapamycin starting material.
Temsirolimus (CCI-779), an mTOR kinase Inhibitor of formula (I) is an antineoplastic agent indicated for the treatment of advanced renal cell carcinoma.Temsirolimus is a Rapamycin 42 ester with [3-hydroxy-2-(hydroxymethyl)-2-methylpropanoic acid and was first disclosed by Skotnicki et al in US Patent No. 5,362,718.
Figure imgf000003_0001
Several processes for the preparation of Temsirolimus have been reported in the literature such as those described in US 5,362,718; US 6,277,983 and US 7, 153,957.
US Patent No 5,362,718 discloses a process for the preparation of different rapamycin 42 esters including Temsirolimus as per the scheme given below (Scheme-I).
Figure imgf000004_0001
Scheme-I: Synthesis of Temsirolimus as disclosed in US Patent No. 5,362,718
The process is non-regioselective and hence results in 31-estehfied rapamycin, 31 , 42 diesterified rapamycin and unreacted rapamycin along with the desired rapamycin-42 ester.
US Patent No. 6,277,983 reports a process for the preparation of Temsirolimus by using 31 , 42 bis silyl intermediates as per the scheme shown below (Scheme-ll).
Figure imgf000005_0001
Scheme-ll: Synthesis of Temsirolimus as disclosed in US Patent No. 6,277,983 US Patent No. 7, 153,957 reports a process for the preparation of Temsirolimusby using boronate intermediate as per the scheme shown below (Scheme-Ill).
Figure imgf000006_0001
Scheme-Ill: Synthesis of Temsirolimus as disclosed in US Patent No. 7, 153,957
Temsirolimus synthesis by Sirolimus (sirolimus, also known as rapamycin Rapamycin) esterification from. Sirolimus is from the soil bacterium Streptomyces hygroscopicus isolated metabolites.Sirolimus 31 and 42 have two alcohol, but 42 slightly smaller steric hindrance. Protected with trimethylsilyl 31 and 42 of the secondary alcohol to give intermediate 1 , 42 for selective removal of sulfuric acid trimethylsilyl obtain 2 , 2 with an acid chloride 3 and a carboxylic acid4 formed by esterification of acid anhydride reaction of 5 under acidic conditions after removal of the 31-bit trimethylsilyl get 6 , 6 with an alcohol 7 boronate protection is removed Temsirolimus. This synthetic route as 31 and 42 to protect the hydroxyl group appear more cumbersome. Later, the development of an enzyme-catalyzed synthesis route (OL2005, 3945). Lipase PS "Amano" (Burkholderia cepacia) of the catalyst, sirolimus and ester 8 reaction of compound 9 .Good selectivity for the enzyme, so that the esterification reaction occurs only in 42, and slightly larger steric hindrance is no response 31. 9 with sulfuric acid for removal of protection is acetonide Temsirolimus.
Temsirolimus-natural product-derived anticancer drugs
Temsirolimus-natural product-derived anticancer drugs
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SYNTHESIS
Example 11
Rapamycin 42-ester with 2.2-bis-(hydroxymethyl)propionic acid
A solution of the product of Example 10 (2.8 g, 2.65 mmol) in 50 mL THF and
25 mL IN HCl was stirred at room temperature for 4 h. The mixture was diluted with water and extracted three times with EtOAc. The combined organic phases were washed with saturated NaHCO3 solution, saturated NaCl solution, dried over MgSO4, filtered and evaporated to a yellow oily solid. Purification by flash chromatography (3X with EtOAc) afforded the title compound (1.6 g, 59 %).
(-)FAB-MS mlz 1029.6 (M-), 590.4 (southern fragment), 437.3 (northern fragment). !H NMR (400 MHz, d-6 DMSO) δ 4.5 (m, 1 H, C(42)H), 3.45 (s, 4 H), 1.04 (s, 3 H).
*3C NMR (100.6 MHz, d-6 DMSO) δ 174.2, 63.7, 63.6, 49.9, 16.8.
Example 10 Rapamycin 42-ester with 2.2.5-trimethyl.1.3_dioxane-5-carboxyric acid
To a solution of the 2,2-bis(hydroxymethyl)propionic acid isopropylidene ketal (1.041 g, 5.98 mmol) (prepared according to the procedure of Bruice, J. Am. Chem. Soc. 89: 3568 (1967)) and triethylamine (0.83 mL, 5.98 mmol) in 20 mL anhydrous THF at 0 °C under nitrogen was added 2, 4, 6-trichlorobenzoyl chloride (0.93 mL, 5.98 mmol) and the resultant white suspension was stirred 5 h at room temperature. The precipitate was removed by vacuum filtration, rinsing the flask and filter cake with an additional 10 mL dry THF. The filtrate was concentrated by rotary evaporation to a white solid. The residue was dissolved in 20 mL dry benzene, then rapamycin (5.47 g, 5.98 mmol) and DMAP (0.731 g, 5.98 mmol) were added. After stirring overnight at room temperature, the mixture was diluted with EtOAc, washed with H2O and saturated NaCl (aq), dried over MgSO4, filtered and evaporated to a yellow oil. Flash chromatography (5X with 60% EtOAc-hexane) afforded the title compound (2.2 g, 34 %) as a white solid.
(-)FAB-MS mlz 1069.5 (M-), 590.3 (southern fragment), 477.2 (northern fragment). -H NMR (400 MHz, d-6 DMSO) δ 4.57 (m, 1 H, C(42)H), 4.02 (d, 2 H), 3.60 (d, 2 H), 1.34 (s, 3 H), 1.24 (s, 3 H), 1.06 (s, 3 H). 1 C NMR (100.6 MHz, d-6 DMSO) δ 173.2, 99.0, 65.0, 22.2, 18.1.
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SYNTHESIS
This scheme
Figure US07153957-20061226-C00004
Figure US07153957-20061226-C00005
Figure US07153957-20061226-C00006
Preparation of 5-Methyl-2-phenyl-1,3,2-dioxaborinane-5-carboxylic acid, [A]
To a suspension of 2,2-bis(hydroxymethyl)propionic acid (131 g, 0.98 mole) in tetrahydrofuran (500 ml) was added a solution of phenylboronic acid (122 g, 1.0 mole) in tetrahydrofuran (500 ml). The mixture was stirred for 3 h and toluene (1.0 L) was added. Water was removed by azeotropic distillation with toluene. Heptanes (500 ml) was added to the precipitated product, heated to reflux and cooled. The mixture was filtered and washed with heptanes (2×300 ml). The solids were dried under vacuum at 70–75° C. until constant weight to give 94% yield. 1H NMR: δ (DMSO-d6) 7.65 (d, 2H, Ar), 7.40 (m, 3H, Ar), 4.35 (d, 2H, CH2), 3.92 (d, 2H, CH2), 1.17 (s, 3H, CH3)
Preparation of Rapamycin 42-ester with 5-methyl-2-phenyl-1,3,2-dioxaborinane-5-carboxylic acid, [B]
As described in U.S. Pat. No. 6,277,983 (2001) a 3 L flask was charged with rapamycin (100 g, 0.104 mole) and dissolved in ethyl acetate (1.50 L). The solution was cooled to 5–10° C. Imidazole (30 g, 0.44 moles, 4.23 eq.) was added and dissolved. Under nitrogen protection, trimethylsilyl chloride (44 g, 0.405 mole, 4.0 eq.) was added over 30–40 min while maintaining the temperature at 0–5° C. during the addition. The mixture was held for a minimum of 0.5 h. The reaction was monitored by TLC (30:70 acetone:heptane eluent). The reaction was complete when all of the rapamycin was consumed.
Two to three drops of the reaction mixture were removed and retained as a 31,42-bis(trimethylsilyl) rapamycin reference standard. 0.5 N Sulfuric acid (300 mL) was added to the 3 L flask over 0.5 h maintaining the temperature 0–5° C. The mixture was stirred vigorously and held for 5 h. The reaction was monitored by thin layer chromatography (TLC) (30:70 acetone:heptane eluent). The reaction was complete when essentially no 31,42-bis-(trimethylsilyl) rapamycin was present. The layers were separated and the lower aqueous layer was back extracted with ethyl acetate (500 mL). The combined organic layers were washed with saturated brine (500 mL) and saturated sodium bicarbonate (2×200 mL) until pH 8 was obtained. The organic layer was washed with water (2×500 mL) and brine (500 ml) until pH 6 to 7 was obtained. The solution was dried over magnesium sulfate (100 g) for 30 min, filtered into a 2 L flask and concentrated to a volume of 135 ml. Ethyl acetate (500 ml) was added and concentrated to a volume of 135 ml. The water chase was repeated once more with ethyl acetate (500 ml). Methylene chloride (300 ml) was added and the solution held until needed in the next step.
A 3 L flask equipped with mechanical stirrer was charged with compound [A] (75 g, 0.341 mole) in methylene chloride (400 mL). Diisopropylethylamine (66.1 g, 0.51 mole) was added dropwise over 20 mins and rinsed with methylene chloride (25 mL). 2,4,6-Trichlorobenzoyl chloride (80 g, 0.328 mole) was added and rinsed with methylene chloride (25 mL). The mixture was held at 0–5° C. for 4 h, and cooled to −10±5° C.
The solution of 31-trimethylsilyl rapamycin was added to the 3 L flask containing the mixed anhydride, and rinsed with methylene chloride (25 mL). A solution of dimethylamino pyridine (48.5 g, 0.397 mole) in methylene chloride (150 mL) was prepared, added over 1.5 h, maintaining the temperature <−8° C., and rinsed with methylene chloride (25 mL). The mixture was held for 12 h at −11 to −5° C. The reaction mixture was quenched with 1 N sulfuric acid (600 ml) keeping the temperature <10° C. The mixture was stirred and held for 30 mins. The pH of the upper aqueous layer was ≦2. The layers were separated, and the lower organic layers washed with brine (450 ml), saturated sodium bicarbonate (500 mL) until pH ≧8. The organic layer was washed with water (450 ml) until pH 6–7 was obtained. The solution was concentrated, acetone (250 ml) added and concentrated. This was repeated with another portion of acetone (250 ml) and concentrated.
The solution was diluted with acetone. 0.5 N Sulfuric acid (500 ml) was added dropwise over 30 mins keeping the pot temperature 0–5° C. The mixture was held for a minimum of 5 h, during which time, the product precipitated out of solution. Aqueous sodium bicarbonate (30 g in 375 ml water) was added dropwise over 30 minutes keeping the pot temperature 0 to 5° C.; the mixture was held for a minimum of 30 minutes. Acetic acid (25 ml) was added until pH was 5–6 keeping the pot temperature <10° C. The mixture was warmed to room temperature and held for 16 h. The solid product was filtered and washed with water (2×100 ml) followed by 1:1 acetone:water (2×100 ml). The cake was purified in acetone (375 ml) to give 65 g (58% overall from rapamycin) of product [B]. LC/MS: using an electrospray interface in the positive ion mode afforded the molecular ion [M+Na]=1138.5 atomic mass units (amu).
Preparation of Rapamycin 42-ester with 2,2-bis(hydroxymethyl)-propionic acid, [C]
Compound [B] (200 g, 0.179 mole), was dissolved in tetrahydrofuran (600 ml), 2-methyl-2,4-pentanediol (42.3 g, 0.358 mole, 2.0 eq.) was added and the mixture stirred for a minimum of 3 h. The reaction mixture was concentrated to a foam. Diethyl ether (1.0 L) was added and the mixture stirred for 2 h. Heptanes (1.0 L) was added dropwise over 1 h and the mixture stirred for 2 h. The mixture was filtered and the solid product washed with heptanes (500 ml). The solids were re-dissolved in acetone (400 ml), re-treated with 2-methyl-2,4-pentanediol (21.1 g, 0.179 mole, 1 eq.) in acetone (200 ml), clarified through a 0.2 micron cartridge filter, and rinsed with acetone (200 ml). The solution was concentrated to a foam, diethyl ether (1.0 L), pre-filtered through a 0.2 micron cartridge filter, was added and the mixture stirred for 2 h. The mixture was co-precipitated by adding pre-filtered heptanes (1.0 L). The precipitated solids were filtered and washed with ether:heptane (2×500 ml). The solids were dried (55 to 60° C., 10 mm Hg, minimum 24 h) to give 159 g (86%) of product [C]. LC/MS: using APCl in the positive ion mode afforded the molecular ion [M+NH4]=1047.0 amu. The 1H NMR of the product (CCl-779) was identical to the product described in example 11 of U.S. Pat. No. 5,362,718 (1994).
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Synthesis
Example 1 - Synthesis of Proline CCI-779
Figure imgf000019_0001
This example describes a method for the synthesis of the proline analog of CCI- 779, which is illustrated in the scheme provided above.
A.
Preparation of 31, 42-Bis (trimethylsilyl) proline rapamycin (Compound B)
A 3 -neck 50 mL flask was charged with proline rapamycin (compound A in the scheme) (1.47 g, 1.63 mmol), imidazole (0.45 g, 6.6 mmol, 4 eq.) and ethyl acetate (22.5 mL). The magnetically stirred mixture became cloudy. The mixture was cooled to 0-5°C. Under nitrogen protection, trimethylsilyl chloride (0.62 g, 5.7 mmol, 3.5 eq.) was added over 0.5 h via syringe while maintaining the temperature at 0-5°C during the addition. The syringe was rinsed with 2.5 ml ethyl acetate and the mixture held for 0.75 hours (0.75 h), whereupon a white precipitate was formed. The reaction was monitored by thin layer chromatography (TLC) (30:70 acetone :heptane eluent). The TLC sample was prepared by quenching 3-4 drops of reaction mixture into 0.25 mL saturated sodium bicarbonate and 10 drops ethyl acetate. The mixture was shaken and allowed to settle. The upper organic layer was spotted against the starting material (proline rapamycin). The reaction was complete when no more starting material was present.
B.
Preparation of 31 -trimethylsilyl proline rapamycin, Compound E
When the above reaction was complete, 2-3 drops of the reaction mixture was removed and retained for the following step as the 31,42-bis(trimethylsilyl) proline rapamycin reference standard. To the 50 ml flask was added 0.5 N sulfuric acid (4.5 mL) over 0.5 h maintaining the temperature at 0-5 °C. The mixture became less cloudy. The mixture was held for 2.5 h and was monitored by thin layer chromatography (TLC, 30:70 acetone:heptane eluent). The TLC sample was prepared by quenching 3-4 drops of reaction mixture into 0.25 mL saturated sodium bicarbonate and 10 drops ethyl acetate. The reaction aliquot was shaken and allowed to settle. The upper organic layer was spotted against the 31 ,42-bis(trimethylsilyl) proline rapamycin reference. The reaction was complete when essentially no 31,42-bis(trimethylsilyl) proline rapamycin was present. Ethyl acetate (5 mL) was added and the layers separated. The lower aqueous layer is extracted with ethyl acetate (7.5 mL). The combined organic layers were washed with brine (7.5 mL), by washing with saturated sodium bicarbonate (6 mL) followed by washing water (3 x 7.5 mL), in that order. The pH of the last water wash was 6-7. The organic layer was washed again with brine (7.5 mL) and dried over sodium sulfate (4 g) for 20 min. The mixture was filtered into a 250 mL flask and concentrated to dryness.
The solid was dried at room temperature under high vacuum (10 mmHg or less) for 20 h.
Weight = 1.51 g of an off-white foam.
C.
Preparation of Intermediate, Compound F:
A 3 -neck 100 mL flask equipped with mechanical stirrer was charged with
2,2,5-trimethyl[l,3-dioxane]-5-carboxylic acid, Compound C (0.63 g, 3.6 mmol) in methylene chloride (7.5 mL). Dusopropylethylamine (0.77 g, 5.9 mmol) was added, followed by a rinse with methylene chloride (1 mL). 2,4,6-Trichlorobenzoyl chloride (0.85 g, 3.5 mmol) was added, followed by a rinse with methylene chloride (1.5 mL).
The mixture was held at room temperature for 4.5 h. The solution was cooled to -12 ±
2°C. 31 -Trimethylsilyl proline rapamycin, compound E, (1.51 g) in methylene chloride (8 mL) was dissolved and added to the 100 mL flask. Methylene chloride (2 mL) was added as a rinse. A solution of dimethylamino pyridine (DMAP) (0.77 g, 6.8 mmol) in methylene chloride (3 mL) was prepared and added to the 100 mL flask over
2.5 h maintaining the temperature -12 ± 2 °C. Methylene chloride (1 mL) was added as a rinse. The mixture was held for 16 h and was monitored by HPLC by quenching 3-4 drops of reaction mixture into 0.25 mL water and 0.2 mL ethyl acetate. The HPLC sample was prepared by withdrawing 2 drops of the upper organic layer, blowdrying the sample under nitrogen in an HPLC vial and redissolving using the mobile phase.
HPLC column : CSC Hypersil ODS / BDS 5 μm.
Mobile phase : 68.5 % dioxane:water + 0.01M KH2P04
Wavelength : λ = 280 nm Flow rate : 1 mL / min
Time : 60 min
Retention times : Compound E ~14.0-14.5 min Compound F -33.4-33.8 min
The reaction was complete when < 0.5% of starting material was present. The reaction mixture was quenched with water (6 mL). Methylene chloride (10 mL) was added and the layers separated. The aqueous layer was extracted with methylene chloride (10 mL). The combined organic layers were washed with 0.5 N sulfuric acid (12 mL), brine (10 mL), saturated sodium bicarbonate (6 mL), and water (3 x 10 mL) in that order. The pH of the last water wash was 6-7. The clear yellow solution was concentrated to a foam. The solid was dried at room temperature under high vacuum (10 mmHg or less) for 24 h. Weight = 1.88 g of a yellow foam.
D.
Preparation of crude proline CCI-779
A 1-neck 50 mL flask equipped with mechanical stirrer was charged with Compound F in THF (18.8 mL, 10 vols) and then cooled to 0 - 5 °C (or about -2.5°C). 2 N sulfuric acid (9.4 mL, 5 vols) was added over 2.5 h. After complete addition, the mixture was warmed to 2.5 °C and then held for 45 h. The reaction was monitored by HPLC by quenching 3-4 drops of reaction mixture into 0.25 mL saturated sodium bicarbonate and 0.25 mL ethyl acetate. The HPLC sample was prepared by withdrawing 5 drops of the upper organic layer, blow drying the sample under nitrogen in an HPLC vial and redissolving using the mobile phase.
HPLC column : CSC Hypersil ODS / BDS 5 μm.
Mobile phase : 68.5 % dioxane:water + 0.01M KH2P04 Wavelength : λ= 280 nm Flow rate : 1 mL / min Time : 60 min Retention times Compound F ~33.4-33.8 min Desilylated Compound F ~10.5-11.5 min (intermediate) Proline CCI-779 -5.0-5.5 min The desilylated intermediate of compound F was formed first. The reaction was complete when < 0.5% of the silylated analog remained. Ethyl acetate (27 mL) and brine (7.5 mL) was added and the layers separated. The aqueous layer was extracted with ethyl acetate (10 mL). The combined organic layers were washed with brine (10 mL), saturated sodium bicarbonate (7.5 mL), and water (3 x 7.5 mL) in that order. The pH of the last water wash was 6-7. The mixture was dried over sodium sulfate (5 g) for 30 min, filtered into a 250 L flask and concentrated to dryness. Weight = 1.58 g of a yellow foam.
E.
Chromatographic purification of crude proline CCI-779
A silica gel column (31.6 g, 60 A, 200-400 mesh) (22 cm length x 2.5 cm diameter) was prepared and conditioned with 15:85 acetone:HPLC grade hexane (1 L). The yellow crude proline CCI-779 (1.58 g) in acetone (1.58 mL) was prepared and chromatographed. The column was eluted with the remaining 15:85 acetone :hexane mixture followed by 25:75 acetone:hexane (4 L). The positive fractions were combined and concentrated to dryness. The resulting foam was dried at 35 °C, high vacuum (i.e., 10 mmHg or less) for 24 h. Weight = 1.12 g of a light yellow foam.
F.
Ether treatment of proline CCI-779
A 1 -neck 50 mL flask was charged with proline CCI-779 ( 1.12 g) and dissolved in ether (1.5 mL). The mixture was held for 2 h. The ether was stripped to give a foam. The foam was dried at 35 °C, under high vacuum (10 mmHg or less) for 12 h then at room temperature overnight (12 h). Weight = 1.09 g.
*H NMR (500 and 600 MHz, DMSO-d6) δ 5.45 (H-l), 6.12 (H-2), 6.27 (H-3), 6.41 (H-4), 6.20 (H-5), 3.66 (H-7), 1.14 and 1.86 (H-8), 4.02 (H-9), 1.19 and 1.81 (H-10), 1.52 (H-11), 2.03 (H-12), 3.23 and 3.54 (H-18), 1.76 (H-19), 2.20 and 1.89 (H-21), 4.22 (H-22), 4.87 (H-25), 2.28 and 2.70 (H-26), 3.22 (H-28), 5.11 (H-29), 4.04 (H-31), 4.17 (H-32), 2.25 (H-34), 0.985 and 1.38 (H-35), 2.22 (H-36), 1.76 (H-37), 0.961 and 1.11 (H-38), 1.31 (H-39), 0.726 and 1.90 (H- 40), 3.14 (H-41), 4.46 (H-42), 1.22 and 1.81 (H-43), 0.888 and 1.60 (H-44), 1.60 (H-45), 3.05 (H-46, OCH3), 0.697 (H-47), 6.48 (H-48), 0.821 (H-49), 1.76 (H-50), approx. 5.1- 5.3 (H-51), 3.17 (H-52, OCH3), 0.755 (H-53), 0.966 (H-54), 0.805 (H-55), 3.29 (H-56, OCH3), 3.46 (H-59), 1.01 (H-60), approx. 4.3-4.7 (0-61)
13C NMR (75 MHz, DMSO- d6) δ 139.12 (C-1), 130.53 (C-2), 132.49 (C-3), 127.08 (C-4), 127.21 (C-5), 137.12 (C-6), 81.93 (C-7), 40.40 (C-8), 65.83 (C-9), 29.45 (C-10), 25.87 (C-l l), 34.21 (C-12), 99.25 (C-13), 198.17 (C-15), 165.55 (C-16), 47.01 (C-18), 24.04 (C-19), 28.93 (C-21), 58.50 (C-22), 170.44 (C-23), 73.24 (C-25), 39.96 (C-26), 207.67 (C-27), 44.51 (C-28), 123.92 (C-29), 136.56 (C-30), 75.84 (C-31), 84.86 (C-32), 209.49 (C-33), 40.76 (C-34), 39.20 (C-35), 35.05 (C-36), 32.73 (C-37), 38.42 (C-38), 32.06 (C-39), 36.01 (C-40), 80.12 (C- 41), 75.92 (C-42), 29.25 (C-43), 30.24 (C-44), 10.27 (C-45), 55.48 (C-46, OCH3), 15.46 (C-47), 15.59 (C-49), 14.41 (C-50), 56.56 (C-52, OCH3), 12.67 (C-53), 21.50 (C-54), 14.89 (C-55), 57.27 (C-56, OCH3), 174.22 (C-57), 49.90 (C-58), 63.59 and 63.98 (C-59), 16.82 (C-60). MS [M+NH ] 1033.5, [ESI(+), M+Na+] 1038.7.
Example 3 - Synthesis of CCI-779:
Figure imgf000025_0001
A. Synthesis of CCI-779 via intermediate A Method 1 : A mixture of rapamycin (6 g), vinyl ester I (2 g), lipase PS-C "Amano" II (6 g) in anhydrous TBME (36 mL) was heated at 45 °C under Ar2 for 2 days. The mixture was cooled to room temperature and enzyme was removed by filtration, the filtrate was concentrated, the oily residue was added to heptane while stirring. The batch was then cooled to -15 °C for 2 h, collect the solid on the Buchner funnel and washed with cold heptane, A was obtained as off-white solid, crude yield : 98%.MS (El): 1070 Above crude A (6g), dissolved in n-PrOH (24 mL) cooled to 0 °C with an ice-water bath, to this solution was added aqueous H2S04 (12 mL, 1.2N). The mixture was stirred for 24 h at 0°C and was then added to cold phosphate buffer (300 ml, pH=7.8), collect the solid on a Buchner funnel and washed with DI water and dry under vacuum, silica gel column purification eluting with hexane-acetone furnished CCI-779 as a white solid (5.2 g, 90%). MS (El): 1030 Method 2: A mixture of rapamycin (30.0 g, 32.8 mmol), vinyl ester I (10.0 g, 50 mmol), lipase PS-C "Amano" II (30 g) and molecular sieves (5 A) (10.0 g) in anhydrous TBME (150 mL) was heated at 42-43 °C under Ar2 for 48 hours. THF (100 mL) was added to dissolve the precipitation and the mixture was cooled to room temperature. Enzyme was removed by filtration and washed with THF (200 mL), the filtrate was concentrated to about 60 mL and diluted with THF (320 mL). The solution was then cooled to 0-5 °C, H2S04 (180 mL, 2N) was added dropwise over lh. The mixture was stirred for 48 h at 0-5 °C or until the disappearance of A as monitored by TLC. The mixture was diluted with brine (300 mL) and extracted with EtOAc (three times). The combined organic layer was washed with H20, 5% NaHC03, then brine and dried
(MgS04). Evaporation of solvent gave a light yellowish semi solid which was purified by flash chromatography (hexane/acetone, 2:1) to give CCI-779 as a white solid (30.77 g, 91% for two steps). B. Synthesis of CCI-779 via intermediate B: A mixture of rapamycin (3 g), vinyl ester II (1.2 g), lipase PS-C "Amano" II (5 g) in anhydrous TBME (45 mL) was heated at 45 °C under Ar2 for 60 h. The mixture was cooled to room temperature and enzyme was removed by filtration, the filtrate was concentrated, MeOH (20 mL) was added to the residue and concentrated to dryness. Silica gel column purification of crude eluting with hexane-acetone furnished CCI-779 as a white solid (2.3 g), and recovered rapamycin (0.81 g). The yield is 93% based on the recovered rapamycin.
proline analog of CCI-779 (proline-rapamycin42-ester with 2,2-bis(hydroxymethyl)propionic acid or proline-CCI-779) and methods of synthesizing same. Proline-CCI-779 is an active drug substance useful in oncology and other associated indications (immunosuppression, anti-inflammatory, anti-proliferation and anti-tumor). In one aspect, the synthesis of proline-CCI-779 is accomplished through bis- silylation of proline rapamycin, mono-de-protecting 31 ,42-bis-trimethylsilyl proline rapamycin, and acylating the mono-silyl proline rapamycin followed by hydrolysis. In another aspect, the invention provides a two-step enzymatic process involving a regiospecific acylation of rapamycin, using a microbial lipase and an activated ester derivative of 2,2-bis(hydroxymethyl)propionic acid in an organic solvent, followed by deprotection to give CCI-779.
Example 4 - Synthesis of Proline-CCI-779 The enzymatic procedure of the invention can also be applied to the synthesis of proline CCI-779 from proline-rapamycin under essentially the same conditions as described in Example 2, procedure A for the synthesis of CCI-779 from rapamycin.
Figure imgf000027_0001
proline-rapamycin proline-CCI-779
......................
more info added for readers
synthesis of CCI-779 or Proline CCI-779 (Temsirolimus) which is useful as an antineoplastic agent having the structure
Figure US08258299-20120904-C00001
It is stated to be effective in multiple applications, including inhibition of tumor growth, the treatment for multiple sclerosis and rheumatoid arthritis.
2. The Prior Arts

U.S. Pat. No. 7,202,256 disclosed methods for the synthesis of CCI-779 (Temsirolimus), providing two-step enzymatic process involving regiospecific acylation of rapamycin, using a microbial lipase and an activated ester derivative of 2,2-bis(hydroxymethyl)propionic acid in an organic solvent, followed by deprotection to obtain the CCI-779 (as shown in scheme 1). A number of drawbacks of the synthesis route depicted in scheme 1 are high-priced PdCland poisonous trimethylboroxine.
Figure US08258299-20120904-C00002
Figure US08258299-20120904-C00003
A selective synthesis of 42-monoacylated product was previously conducted by reacting rapamycin 31,42-bis-silyl ether, and then the 42-sily ether protection group is selectively removed to provide rapamycin-OH-31-sily ether (U.S. Pat. No. 5,563,145). In addition, a regioselective process for the preparation of CCI-779 is also described in U.S. Pat. No. 6,277,983 (Scheme2). First, rapamycin (compound 4b) is treated with excess chlorotrimethylsilane to form rapamycin31,42-bis-trimethylsilyl ether (compound 5), and then 42-trimethylsilyl ether protection group is selectively removed in mild acid to provide rapamycin 42-OH-31-trimethylsilyl ether (compound 6). This free 42-OH was then acylated with 2,4,6-trichlorobenzyl mixed anhydride of 2,2,5-trimethyl[1,3-dioxane]-5-carboxylic acid (compound 7) at −15° C. for 16 h to give rapamycin 31-trimethylsilyl ether 42-ester (compound 8). Following treatment with mild acid for a certain period, CCI-779 can be isolated. 2,4,6-trichlorobenzyl chloride is irritant, moisture sensitive and costly.
Figure US08258299-20120904-C00004
Further, as below-depicted in Scheme 3, U.S. Pat. No. 7,153,957 disclose another method for the CCI-779. It can be prepared by the acylation of 31-silyl ether of rapamycin with the anhydride derived from the 2-phenylboronate acid to give rapamycin 31-silyl ether, 42-boronate. Thereafter, it is hydrolyzed under mild acid condition to form rapamycin 42-ester boronate. After being treated with a suitable diol, CCI-779 was obtained (Scheme 3). Mixed anhydride is not satisfactory for commercial scale synthesis because it can be kept stable only for 48 hr at −5˜0° C., not durable for longer time.
synthesis ofTemsirolimus in a more economic way.
Figure US08258299-20120904-C00005


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TEMSIROLIMUS


PAPERS

CCI-779
Drugs Fut 2002, 27(1): 7

Organic Letters, 2005 ,  vol. 7,  18  pg. 3945 - 3948   seenmr




PATENTS
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US72022564-11-2007Proline CCI-779, production of and uses therefor, and two-step enzymatic synthesis of proline CCI-779 and CCI-779
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US70748047-12-2006CCI-779 Isomer C
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3 EVEROLIMUS

Afinitor (everolimus)

40-O-(2-hydroxyethyl)-rapamycin
Company: Novartis
Approval Status: Approved July 2012
Treatment Area: hormone receptor-positive, HER2-negative breast cancer
Everolimus is a derivative of Rapamycin (sirolimus), and works similarly to Rapamycin as an mTOR (mammalian target of rapamycin) inhibitor. It is currently used as an immunosuppressant to prevent rejection of organ transplants. In a similar fashion to other mTOR inhibitors Everolimus' effect is solely on the mTORC1 protein and not on the mTORC2 protein.
Also known as: Afinitor, Certican, Zortress, SDZ-RAD, RAD001, Everolimus [USAN], 42-O-(2-Hydroxyethyl)rapamycin, RAD 001
Molecular Formula: C53H83NO14   Molecular Weight: 958.22442
159351-69-6  CAS NO
BRANDS
AfinitorNovartis
CerticanNovartis
VOTUBIANovartis
ZortressNovartis

Afinitor (everolimus), an inhibitor of mTOR (mammalian target of rapamycin), is an antineoplastic agent.

Afinitor is specifically approved for the treatment of postmenopausal women with advanced hormone receptor-positive, HER2-negative breast cancer (advanced HR+ BC) in combination with exemestane, after failure of treatment with letrozole or anastrozole.
Afinitor is supplied as a tablet for oral administration. The recommended dose of Afinitor for breast cancer is 10 mg, to be taken once daily, at the same time every day, either consistently with food or consistently without food.
FDA Approval
The FDA approval of Afinitor for the treatment of advanced hormone receptor-positive, HER2-negative breast cancer was based on a randomized, double-blind, multicenter study in 724 postmenopausal women with estrogen receptor-positive, HER 2/neu-negative advanced breast cancer with recurrence or progression following prior therapy with letrozole or anastrozole.
Everolimus is indicated for the treatment of postmenopausal women with advanced hormone receptor-positive, HER2-negative breast cancer (advanced HR+ BC) in combination with exemestane, after failure of treatment with letrozole or anastrozole. Indicated for the treatment of adult patients with progressive neuroendocrine tumors of pancreatic origin (PNET) with unresectable, locally advanced or metastatic disease. Indicated for the treatment of adult patients with advanced renal cell carcinoma (RCC) after failure of treatment with sunitinib or sorafenib. Indicated for the treatment of adult patients with renal angiomyolipoma and tuberous sclerosis complex (TSC), not requiring immediate surgery. Indicated in pediatric and adult patients with tuberous sclerosis complex (TSC) for the treatment of subependymal giant cell astrocytoma (SEGA) that requires therapeutic intervention but cannot be curatively resected.
Everolimus (RAD-001) is the 40-O-(2-hydroxyethyl) derivative of sirolimus and works similarly to sirolimus as an inhibitor of mammalian target of rapamycin (mTOR).
It is currently used as an immunosuppressant to prevent rejection of organ transplants and treatment of renal cell cancer and other tumours. Much research has also been conducted on everolimus and other mTOR inhibitors for use in a number of cancers.
It is marketed by Novartis under the tradenames Zortress (USA) and Certican (Europe and other countries) in transplantation medicine, and Afinitor in oncology.
EVEROLIMUS
AFINITOR (everolimus), an inhibitor of mTOR, is an antineoplastic agent.
The chemical name of everolimus is (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18- dihydroxy-12-{(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl}-19,30-dimethoxy15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-aza-tricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20pentaone.
The molecular formula is C53H83NO14 and the molecular weight is 958.2. The structural formula is:
AFINITOR (everolimus) Structural Formula Illustration
AFINITOR Tablets are supplied for oral administration and contain 2.5 mg, 5 mg, 7.5 mg, or 10 mg of everolimus. The tablets also contain anhydrous lactose, butylated hydroxytoluene, crospovidone, hypromellose, lactose monohydrate, and magnesium stearate as inactive ingredients.
AFINITOR DISPERZ (everolimus tablets for oral suspension) is supplied for oral administration and contains 2 mg, 3 mg, or 5 mg of everolimus. The tablets for oral suspension also contain butylated hydroxytoluene, colloidal silicon dioxide, crospovidone, hypromellose, lactose monohydrate, magnesium stearate, mannitol, and microcrystalline cellulose as inactive ingredients.
Links
  1. R.N Formica Jra, K.M Lorberb, A.L Friedmanb, M.J Biaa, F Lakkisa, J.D Smitha, M.I Lorber (March 2004). “The evolving experience using everolimus in clinical transplantation”. Elsevier 36 (2): S495–S499.
  2.  “Afinitor approved in US as first treatment for patients with advanced kidney cancer after failure of either sunitinib or sorafenib” (Press release). Novartis. 2009-03-30. Retrieved April 6, 2009.
  3. “Novartis receives US FDA approval for Zortress (everolimus) to prevent organ rejection in adult kidney transplant recipients” (Press release). Novartis. 2010-04-22. Retrieved April 26, 2010.
  4. “Novartis’ Afinitor Cleared by FDA for Treating SEGA Tumors in Tuberous Sclerosis”. 1 Nov 2010.
  5. http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm254350.htm
  6. “US FDA approves Novartis drug Afinitor for breast cancer”. 20 Jul 2012.
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...........................................
Rapamycin is a known macrolide antibiotic produced by Streptomvces hvgroscopicus. having the structure depicted in Formula A:
Figure imgf000003_0001
See, e.g., McAlpine, J.B., et al., J. Antibiotics (1991) 44: 688; Schreiber, S.L., et al., J. Am. Chem. Soc. (1991) J_13: 7433'- US Patent No. 3 929 992. Rapamycin is an extremely potent immunosuppressant and has also been shown to have antitumor and antifungal activity. Its utility as a pharmaceutical, however, is restricted by its very low and variable bioavailabiiity as well as its high toxicity. Moreover, rapamycin is highly insoluble, making it difficult to formulate stable galenic compositions.
Everolimus, 40-O-(2-hydroxyethyl)-rapamycin of formula (1) is a synthetic derivative of rapamycin (sirolimus) of formula (2), which is produced by a certain bacteria strain and is also pharmaceutically active.
Figure imgf000002_0002
(1)                                                                                                               (2)
Everolimus is marketed under the brand name Certican for the prevention of rejection episodes following heart and kidney transplantation, and under the brand name Afinitor for treatment of advanced kidney cancer.
Due to its complicated macrolide chemical structure, everolimus is, similarly as the parent rapamycin, an extremely unstable compound. It is sensitive, in particular, towards oxidation, including aerial oxidation. It is also unstable at temperatures higher than 25°C and at alkaline pH.
Everolimus and a process of making it have been disclosed in WO 94/09010
Synthesis
Alkylation of rapamycin (I) with 2-(tert-butyldimethylsilyloxy)ethyl triflate (II) by means of 2,6-lutidine in hot toluene gives the silylated target compound (III), which is deprotected by means of 1N HCl in methanol (1). (Scheme 21042401a) Manufacturer Novartis AG (CH). References 1. Cottens, S., Sedrani, R. (Sandoz-Refindungen VmbH; Sandoz-Patent GmbH; Sandoz Ltd.). O-Alkylated rapamycin derivatives and their use, particularly as immunosuppressants. EP 663916, EP 867438, JP 96502266, US 5665772, WO 9409010.EP 0663916; EP 0867438; JP 1996502266; JP 1999240884; US 5665772; WO 9409010
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SYNTHESIS
(US 5,665,772, EP 663916). The process principle is shown in the scheme below, wherein the abbreviation RAP-OH has been used as an abbreviation for the rapamycin structure of formula (2) above, L is a leaving group and P is a trisubstituted silyl group serving as a OH- protective group.
RAP-OH + L-CH2-CH2-0-P — --> RAP-O-CH2-CH2-O-P — - > RAP-O-CH2-CH2-OH
(2)                                                 (4)                                                                 (1)
Specifically, the L- group is a trifluoromethanesulfonate (triflate) group and the protective group P- is typically a tert-butyldimethylsilyloxy- group. Accordingly, the known useful reagent within the above general formula (3) for making everolimus from rapamycin is 2-(tert-butyldimethylsilyloxy)ethyl triflate of formula (3 A):
Figure imgf000003_0001
According to a known synthetic procedure disclosed in Example 8 of WO 94/09010 and in Example 1 of US application 2003/0125800, rapamycin (2) reacts in hot toluene and in the presence of 2,6-lutidine with a molar excess of the compound (3 A), which is charged in several portions, to form the t-butyldimethylsilyl-protected everolimus (4A). This compound is isolated and deprotected by means of IN aqueous HC1 in methanol. Crude everolimus is then purified by column chromatography. Yields were not reported.
Figure imgf000004_0001
(2)                                       (3A)                              (4A)                                (1)
In an article of Moenius et al. (J. Labelled Cpd. Radiopharm. 43, 113-120 (2000)), which used the above process for making C14-labelled and tritiated everolimus, a diphenyl- tert.butylsilyloxy -protective group was used as the alkylation agent of formula (3B).
Figure imgf000004_0002
Only 8% yield of the corresponding compound (4B)
Figure imgf000004_0003
and 21% yield of the compound (1) have been reported.
Little is known about the compounds of the general formula (3) and methods of their preparation. The synthesis of the compound (3 A) was disclosed in Example 1 of US application 2003/0125800. It should be noted that specification of the reaction solvent in the key step B of this synthesis was omitted in the disclosure; however, the data about isolation of the product allow for estimation that such solvent is dichloromethane. Similarly also a second article of Moenius et al. (J. Labelled Cpd. Radiopharm.42, 29-41 (1999)) teaches that dichloromethane is the solvent in the reaction.
It appears that the compounds of formula (3) are very reactive, and thus also very unstable compounds. This is reflected by the fact that the yields of the reaction with rapamycine are very low and the compound (3) is charged in high molar extent. Methods how to monitor the reactivity and/or improve the stability of compounds of general formula (3), however, do not exist.
Thus, it would be useful to improve both processes of making compounds of formula (3) and, as well, processes of their application in chemical synthesis.
xample 6: 40-O-[2-((2,3-dimethylbut-2-yl)dimethylsilyloxy)ethyl]rapamycin
In a 100 mL flask, Rapamycin (6 g, 6.56 mmol) was dissolved in dimethoxyethane (4.2 ml) and toluene (24 ml) to give a white suspension and the temperature was raised to 70°C. After 20 min, N,N-diisopropylethylamine (4.56 ml, 27.6 mmol) and 2-((2,3-dimethylbutan-2- yl)dimethylsilyloxy)ethyl trifluoromethanesulfonate (8.83 g, 26.3 mmol) were added in 2 portions with a 2 hr interval at 70°C. The mixture was stirred overnight at room temperature, then diluted with EtOAc (40 ml) and washed with sat. NaHC03 (30 ml) and brine (30 ml). The organic layer was dried with Na2S04, filtered and concentrated. The cmde product was chromatographed on a silica gel column (EtOAc/heptane 1/1 ; yield 4.47 g).
Example 7: 40-O-(2-hydroxyethyl)-rapamycin [everolimus]
In a 100 mL flask, 40-O-[2-((2,3-dimethylbut-2-yl)dimethylsilyloxy)ethyl]rapamycin (4.47 g, 4.06 mmol) was dissolved in methanol (20 ml) to give a colorless solution. At 0°C, IN aqueous hydrochloric acid (2.0 ml, 2.0 mmol) was added and the mixture was stirred for 90 min. The reaction was followed by TLC (ethyl acetate/n-heptane 3 :2) and HPLC. Then 20 ml of saturated aqueous NaHC03 were added, followed by 20 ml of brine and 80 ml of ethyl acetate. The phases were separated and the organic layer was washed with saturated aqueous NaCl until pH 6/7. The organic layer was dried by Na2S04, filtered and concentrated to yield 3.3 g of the product.
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SYNTHESIS
Example 8: 40-O-(2-Hydroxy)ethyl-rapamycin
a) 40-O-[2-(t-Butyldimethylsilyl)oxy]ethyl-rapamycin
A solution of 9.14 g (10 mmol) of rapamycin and 4.70 mL (40 mmol) of 2,6-lutidine in 30 mL of toluene is warmed to 60°C and a solution of 6.17 g (20 mmol) of 2-(t-butyldimethylsilyl)oxyethyl triflate and 2.35 mL (20 mmol) of 2,6-lutidine in 20 mL of toluene is added. This mixture is stirred for 1.5h. Then two batches of a solution of 3.08 g (10 mmol) of triflate and 1.2 mL (10 mmol) of 2,6-lutidine in 10 mL of toluene are added in a 1.5h interval. After addition of the last batch, stirring is continued at 60°C for 2h and the resulting brown suspension is filtered. The filtrate is diluted with ethyl acetate and washed with aq. sodium bicarbonate and brine. The organic solution is dried over anhydrous sodium sulfate, filtered and concentrated. The residue is purified by column chromatography on silica gel (40:60 hexane-ethyl acetate) to afford 40-O-[2-(t-butyldimethylsilyl)oxy]ethyl-rapamycin as a white solid: 1H NMR (CDCl3) δ 0.06 (6H, s), 0.72 (1H, dd), 0.90 (9H, s), 1.65 (3H, s), 1.75 (3H, s), 3.02 (1H, m), 3.63 (3H, m), 3.72 (3H, m); MS (FAB) m/z 1094 ([M+Na]+), 1022 ([M-(OCH3+H2O)]+).
b) 40-O-(2-Hydroxy)ethyl-rapamycin
To a stirred, cooled (0°C) solution of 4.5 g (4.2 mmol) of 40-O-[2-(t-butyldimethylsilyl)oxy]ethyl-rapamycin in 20 mL of methanol is added 2 mL of IN HCl. This solution is stirred for 2h and neutralized with aq. sodium bicarbonate. The mixture is extracted with three portions of ethyl acetate. The organic solution is washed with aq.
sodium bicarbonate and brine, dried over anhydrous sodium sulfate, filtered and
concentrated. Purification by column chromatography on silica gel (ethyl acetate) gave the title compound as a white solid:1H NMR (CDCl3) δ 0.72 (1H, dd), 1.65 (3H, s), 1.75 (3H, s), 3.13 (5H, s and m), 3.52-3.91 (8H, m); MS (FAB) m/z 980 ([M+Na]+), 926 ([M-OCH3]+), 908 ([M-(OCH3+H2O)]+), 890 ([M-(OCH3+2H2O)]+), 876 ([M-(2CH3OH+OH)]+), 858 ([M-(OCH3+CH3OH+2H2O)]+).
MBA (rel. IC50) 2.2
IL-6 dep. prol. (rel. IC50) 2.8
MLR (rel. IC50) 3.4
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synthesis
Everolimus (Everolimus) was synthesized by the Sirolimus (sirolimus, also known as rapamycin Rapamycin) ether from. Sirolimus is from the soil bacterium Streptomyces hygroscopicus isolated metabolites. Activation end sirolimus (triflate, Tf) the other end of the protection (t-butyldimethylsilyl, TBS) of ethylene glycol 1 reaction of 2 , because the hydroxyl group 42 hydroxyl site over the 31-bit resistance is small, so the reaction only occurs in 42. Compound 2under acidic conditions TBS protection is removed everolimus.
Everolimus (Everolimus) - natural product derived anticancer drugs

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